Picornaviruses are major etiological agents of human diseases, including the common cold, poliomyelitis, encephalitis, meningitis, myocarditis, and hepatitis. They are the most common causes of viral illnesses worldwide and the most common causes of infections of humans in the developed world. Yet they are among the most genetically simple of all mammalian viruses, harboring small, positive-sense genomic RNAs with critical regulatory elements contained within their long 5' noncoding regions (NCRs). This apparent conundrum can, in part, be explained by the way these viruses hijack host cell functions and modify the cytoplasmic environment to favor viral replication. Picornaviruses have evolved complex mechanisms for key steps in their replication cycles, including IRES-mediated translation initiation, the switch in template use from translation to RNA replication, and the evasion of host anti-viral responses that all involve, directly or indirectly, the genomic 5' NCR. Unraveling these mechanisms is the over-arching theme of the experiments proposed in this renewal application. Using poliovirus, human rhinovirus, and coxsackievirus in cell culture models and an array of in vitro experimental approaches, this application will address the fundamental nature of viral processes that are directed by the 5' NCRs of genomic RNAs by proposing to (i) determine how host protein SRp20 interacts with the 40S ribosomal subunit to mediate formation of poliovirus IRES-mediated translation initiation complexes, (ii) define the steps used by human rhinovirus to switch template usage from translation to viral RNA synthesis, and (iii) determine the mechanism of AUF1 negative regulation of viral infections. Results from the proposed studies will reveal important insights into how the 5' NCRs of picornavirus RNAs control a myriad of interactions with host and viral proteins, all in the name of survival of a genetically-challenged family of RNA viruses. Such insights should be broadly applicable to other positive-strand RNA viruses that replicate in the cytoplasm of infected human cells and may help to identify unique virus-host interfaces to be developed as targets for anti-viral therapeutics.